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Expanded CTG repeats within the DMPK 3' UTR causes severe skeletal muscle wasting in an inducible mouse model for myotonic dystrophy - PubMed

  • ️Tue Jan 01 2008

Expanded CTG repeats within the DMPK 3' UTR causes severe skeletal muscle wasting in an inducible mouse model for myotonic dystrophy

James P Orengo et al. Proc Natl Acad Sci U S A. 2008.

Abstract

Severe skeletal muscle wasting is the most debilitating symptom experienced by individuals with myotonic dystrophy type 1 (DM1). We present a DM1 mouse model with inducible and skeletal muscle-specific expression of large tracts of CTG repeats in the context of DMPK exon 15. These mice recapitulate many findings associated with DM1 skeletal muscle, such as CUG RNA foci with Muscleblind-like 1 (MBNL1) protein colocalization, misregulation of developmentally regulated alternative splicing events, myotonia, characteristic histological abnormalities, and increased CUGBP1 protein levels. Importantly, this DM1 mouse model recapitulates severe muscle wasting, which has not been reported in models in which depletion of MBNL1 is the main feature. Using these mice, we discovered previously undescribed alternative splicing events that are responsive to CUGBP1 and not MBNL, and these events were found to be misregulated in individuals with DM1. Our results indicate that increased CUGBP1 protein levels are associated with DMPK-CUG RNA expression, suggesting a role for CUGBP1-specific splicing or cytoplasmic functions in muscle wasting.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

Bitransgenic animals expressing either 0 or 960 CUG repeats within exon 15 of the DMPK gene. (A) Transgene driven by the CMV enhancer was created with a floxed concatemer of three polyadenylation sites followed by exon 15 of the DMPK gene with either 0 (designated EpA0) or 960 (EpA960) CTG repeats (19). EpA lines transcribe RNAs that terminate at one of the three polyadenylation sites. Cre-mediated recombination induces expression of mRNA containing DMPK exon 15 with [EpA960(R)] or without [EpA0(R)] CUG repeats. (B) Quantitative RT-PCR performed on RNA extracted from gastrocnemius muscle in EpA960/HSA-Cre-ERT2 mice (n = 5 for each group) and EpA0/HSA-Cre-ERT2 mice (n = 3 for each group) before and 4 weeks after tamoxifen administration. GAPDH was used as a standard. Differences in mRNA levels from the recombined allele [EpA0(R) or EpA960(R)] between pre- and posttamoxifen in EpA960/HSA-Cre-ERT2 and EpA0/HSA-Cre-ERT2 mice were statistically significant (*) as assessed by Student's t test, P < 0.05. (C) Expression of mRNA from the recombined allele in different muscle groups (Quad, quadriceps; Gas, gastrocnemius; Sol, soleus; Tri, triceps; Dia, diaphragm) in EpA960/HSA-Cre-ERT2 mice 4 weeks after tamoxifen. There is no statistically significant difference between the five muscle groups using one-way ANOVA, n = 3 for each group.

Fig. 2.
Fig. 2.

EpA960/HSA-Cre-ERT2 (+ tam) mice reproduce the molecular features of DM1. (A) Intranuclear CUG repeat RNA foci with MBNL1 colocalization is not detected in triceps muscle isolated from HSA-Cre-ERT2 (Upper) and is present in EpA960/HSA-Cre-ERT2 (Lower) 4 weeks after tamoxifen administration. All images acquired at ×63 and with the same exposure times for the red and green filters. (Scale bar: 20 μm.) (B) EpA960/HSA-Cre-ERT2 (+ tam) mice display misregulation of developmentally regulated alternative splicing events as seen in DM1. RT-PCR was performed by using primers that flank the alternative exon (

SI Table 1

). The percentage inclusion of alternative exons from three genes is graphically represented. Lanes are as follows: Newborn, WT day 1 newborn limb; HSA-Cre, HSA-Cre-ERT2 no tam; Bi(960), EpA960/HSA-Cre-ERT2 no tam; Bi(960) + 1 wk, EpA960/HSA-Cre-ERT2 1 week posttamoxifen (post tam); Bi (960) + 4 wks, EpA960/HSA-Cre-ERT2 4 weeks post tam; Bi (0) + 4wks, EpA0/HSA-Cre-ERT2 4 weeks post tam. Each bar represents the mean of the three biological replicates with standard deviation, except for the Newborn sample, which represents a pool of >12 animals. One-way ANOVA followed by Tukey–Kramer analysis was used to assign the samples into groups (a, b, or c) for each graph. Animals assigned to one group but not another are statistically different. Gel images are in

SI Fig. 6

. (C) Electromyogram traces from the gastrocnemius muscle of an EpA960/HSA-Cre-ERT2 (+ tam) mouse, showing portions of two distinct, sustained myotonic runs with characteristically audible waxing and waning frequency. Vertical scale, 0.2 mV per division; horizontal scales, 25 ms per division (Upper) and 50 ms per division (Lower).

Fig. 3.
Fig. 3.

EpA960/HSA-Cre-ERT2 mice exhibit severe and progressive skeletal muscle wasting and dysfunction after tamoxifen administration. (A) HSA-Cre-ERT2 (Upper) and EpA960/HSA-Cre-ERT2 (Lower) littermates 4 weeks after tamoxifen administration. (B) From left to right, the hind limbs of age- and sex-matched HSA-Cre-ERT2, EpA0/HSA-Cre-ERT2, and EpA960/HSA-Cre-ERT2 mice 4 weeks after tamoxifen administration. Arrows indicate quadriceps and gastrocnemius muscle bodies. (C) Wasting of the paraspinous muscle determined by MRI from one HSA-Cre-ERT2 (dashed line) and three EpA960/HSA-Cre-ERT2 (solid lines) mice during a 4-week time course after tamoxifen administration. Measurements were normalized to the kidney diameter. (D) Progressive loss of muscle function in EpA960/HSA-Cre-ERT2 mice after tamoxifen administration. All mice were assayed before tamoxifen administration and then at 1 and 4 weeks after tamoxifen by using a graded treadmill protocol (see Materials and Methods). The box plot represents 10 HSA-Cre-ERT2 mice (black diamonds) and 10 EpA960/HSA-Cre-ERT2 mice (blue diamonds). Data points between the upper 75th and lower 25th quartiles are boxed; the horizontal line within the box is the median value. Using the Student t test, there is no statistically significant difference in HSA-Cre-ERT2 mice between the three time points, but there is a statistically significant difference in EpA960/HSA-Cre-ERT2 mice when the pretamoxifen time point is compared with the 1-week- and 4-weeks-after-tamoxifen time points, P < 0.05. (E) EpA960/HSA-Cre-ERT2 (+ tam) mice show a progressive muscle dystrophy by histology. Note the increasing numbers of central nuclei, small basophilic fibers, fiber diameter variation, and fibrosis seen in EpA960/HSA-Cre-ERT2 mice after tamoxifen administration. All images are at ×40 magnification. (Scale Bar: 50 μm.)

Fig. 4.
Fig. 4.

Increased CUGBP1 expression is a potential mechanism for muscle wasting in EpA960/HSA-Cre-ERT2 mice. (A) EpA960/HSA-Cre-ERT2 mice show increased steady state levels of CUGBP1 protein by Western blot analysis. (B) Percentage inclusion of alternative exons in Ank2, Capzb, and Fxr1 in skeletal muscle tissues across several time points after tamoxifen in EpA960/HSA-Cre-ERT2 mice and from two other DM1 mouse models. Each bar is a mean of biologic replicates with standard deviation when n is >1. Lanes: Newborn, day 1 newborn limb (n = 1, sample is pool of 12 animals); HSA-Cre, HSA-Cre-ERT2 no tam (n = 3); Bi (960), EpA960/HSA-Cre-ERT2 no tam (n = 3); Bi (960) + 1 wk, EpA960/HSA-Cre-ERT2 1 week after tam (n = 3); Bi (960) + 4 wks, EpA960/HSA-Cre-ERT2 4 weeks after tam (n = 3); Bi (0) + 4wks, EpA0/HSA-Cre-ERT2 4 weeks after tam (n = 3); WT, wild-type control for Mbnl1Δ3/Δ3 (n = 1); Mbnl1Δ3/Δ3, Mbnl1Δ3/Δ3 (n = 1); HSA(SR), HSASR, similar to HSALR but contains 5 CTG repeats (n = 3); HSA(LR), HSALR (n = 3). Each animal group was compared with HSA-Cre, and any statistically significant differences were determined by Student's t test, P < 0.05 (*). (C) Percentage inclusion of alternative exons in Ank2, Capzb, and Fxr1 in human skeletal muscle samples from normal fetal (white n = 1) and adult (black n = 3) tissue as well as adult DM1 tissue (gray n = 5 or 6). *, The normal adult skeletal muscle sample has a statistically significant difference from both the normal fetal and adult DM1 tissue samples, determined with the Student's t test, P < 0.05. Gel images for B and C are in

SI Figs. 11 and 12

, respectively; information on human samples is located in

SI Table 2

.

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References

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